The proposed project aims at developing a novel therapeutic intervention against chronic hepatitis B, which currently remains a significant public health challenge worldwide. We will explore the development of a group of newly identified compounds that inhibit the formation of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, which is not targeted by current available antiviral medications. HBV cccDNA plays a central role in viral infection establishment and persistence, and is the basis for the failure of current antiviral treatments and virus rebound after the cessation of therapy. Therefore the elimination of cccDNA is the ultimate goal in curing HBV infection. Through our efforts to screen an 80,300 small molecule library using an innovative cell-based cccDNA high throughput assay, two structurally related disubstituted-sulfonamides (DSS) were discovered that have dramatic inhibition of cccDNA formation by interfering with the deproteinization of HBV genomic relaxed circular (rc) DNA, which serves as the intermediate precursor for cccDNA. To determine whether Investigational New Drug (IND)-enabling studies are justified, we plan to optimize these compounds, assess their utility against drug resistant infections and in combination with existing drugs in vitro, determine their in vivo characteristics and efficacy, and further study their mechanism of action. Successful completion of our goals will produce at least one new lead to advanced preclinical studies. PUBLIC HEALTH RELEVANCE: Hepatitis B virus (HBV) infection is a severe public health problem affecting about 350 million individuals worldwide. Chronic hepatitis B patients have a high risk of occurrence of hepatocirrhosis and liver cancer. Currently approved drugs for treatment of chronic hepatitis B, including alpha interferon, and nucleos(t)ide analogues inhibiting the viral polymerase, are limited by poor response, side effects and emergence of drug resistance. In our proposal, we are aiming at the development of a non-classical anti-HBV agent with novel antiviral target. Our research is thus highly related to the public health interest. The accomplishment of proposed research will ultimately lead to the better therapeutic for the management of hepatitis B disease.